Method for Measuring Heptoglobin Level in Blood Serum and Kit Therefor

Abstract

The present invention relates to a method for measuring heptoglobin level in blood serum by using antibodies for α and β subunits of heptoglobin, and a kit for immunodetection of heptoglobin which comprises the antibodies for α and β subunits of heptoglobin for measuring heptoglobin level in blood serum. A method for measuring heptoglobin level in blood serum of the present invention comprises the steps of: reacting blood serum of a subject with an antibody (αh antibody) which binds specifically to α subunit of a dimeric heptoglobin and an antibody (βh antibody) which binds specifically to β subunit of a dimeric heptoglobin; and, measuring level of proteins which react with the αh antibody and βh antibody in blood serum and have a molecular weight of more than 100 kDa. The method for measuring heptoglobin level in blood serum can be practically applied for the early diagnosis of diseases which disrupt erythrocytes, since only the level of normal heptoglobin except for monomelic and degraded hep-toglobins can be specifically measured by the said method.

1 . A method for measuring heptoglobin level in blood serum, which comprises the steps of:
 (i) reacting blood serum of a subject with an antibody (αh antibody) which binds specifically to α subunit of a dimeric heptoglobin and an antibody (βh antibody) which binds specifically to β subunit of a dimeric heptoglobin; and,   (ii) measuring level of proteins which react with the αh antibody and βh antibody in blood serum and have a molecular weight of more than 100 kDa.   
   
   
       2 . The method for measuring heptoglobin level in blood serum of  claim 1 , wherein the level of proteins is measured by Western blot analysis. 
   
   
       3 . The method for measuring heptoglobin level in blood serum of  claim 1 , wherein the level of proteins is measured by Sandwich ELISA method. 
   
   
       4 . The method for measuring heptoglobin level in blood serum of  claim 3 , wherein the level of proteins is measured by using a labeled αh antibody and a non-labeled βh antibody. 
   
   
       5 . The method lee measuring heptoglobin level in blood serum of  claim 3 , wherein the level of proteins is measured by using a non-labeled αh antibody and a labeled βh antibody. 
   
   
       6 . The method for measuring heptoglobin level in blood serum of  claim 4 , wherein the labeling of antibody is done by fluorescent material, radioisotope or enzyme. 
   
   
       7 . The method for measuring heptoglobin level in blood serum of  claim 6 , wherein the fluorescent material is Cy-3, Cy-5, FITC (fluorescein isothiocyanate), GFP (green fluorescent protein), RFP (red fluorescent protein) or Texas Red. 
   
   
       8 . The method for measuring heptoglobin level in blood serum of  claim 6 , wherein the enzyme is HRP (horse raddish peroxidase), alkaline phosphatase, beta-galactosidase or luciferase. 
   
   
       9 . A method for diagnosing an attack of disease which disrupts erythrocyte, which comprises the steps of:
 reacting blood serum of a subject with an antibody (αh antibody) which binds specifically to α subunit of a dimeric heptoglobin and an antibody (βh antibody which binds specifically to β subunit of a dimeric heptoglobin;   (ii) measuring level of proteins which react with the αh antibody and βh antibody in blood serum and have a molecular weight of more than 100 kDa; and,   (iii) comparing the measured protein level with protein level of a normal.   
   
   
       10 . The method for diagnosing an attack of disease which disrupts erythrocyte of  claim 9 , wherein the level of proteins is measured by Western blot analysis. 
   
   
       11 . The method for diagnosing an attack of disease which disrupts erythrocyte of  claim 9 , wherein the level of proteins is measured by Sandwich ELISA method. 
   
   
       12 . The method for diagnosing an attack of disease which disrupts erythrocyte of  claim 11 , wherein the level of proteins is measured by using a labeled αh antibody and a non-labeled βh antibody. 
   
   
       13 . The method for diagnosing an attack of disease which disrupts erythrocyte of  claim 11 , wherein the level of proteins is measured by using a non-labeled αh antibody and a labeled βh antibody. 
   
   
       14 . The method for diagnosing an attack of disease which disrupts erythrocyte of  claim 12 , wherein the labeling of antibody is done by fluorescent material, radioisotope or enzyme. 
   
   
       15 . The method for diagnosing an attack of disease which disrupts erythrocyte of  claim 14 , wherein the fluorescent material is Cy-3, Cy-5, FITC (fluorescein isothiocyanate), GFP (green fluorescent protein), RFP (red fluorescent protein) or Texas Red. 
   
   
       16 . The method for diagnosing an attack of disease which disrupts erythrocyte of  claim 14 , wherein the enzyme is HRP (horse raddish peroxidase), alkaline phosphatase, beta-galactosidase or luciferase. 
   
   
       17 . The method for diagnosing an attack of disease which disrupts erythrocyte of  claim 9 , wherein the disease is malaria. 
   
   
       18 . A kit for immunodetection of heptoglobin which comprises;
 (i) a plate on which an antibody which binds specifically to α subunit or β subunit of heptoglobin is fixed;   (ii) a solution containing an antibody which binds specifically to α subunit or β subunit which is not bound to the antibody on the plate;   (iii) a secondary antibody which binds to the antibody in the solution; and,   (iv) a means for detecting the secondary antibody.   
   
   
       19 . The kit for immunodetection of heptoglobin of  claim 18 , wherein the plate is glass or plastic. 
   
   
       20 . The kit for immunodetection of heptoglobin of  claim 18 , wherein the secondary antibody is labeled with biotin- or peroxidase-labeled rabbit-goat serum. 
   
   
       21 . The kit for immunodetection of heptoglobin of  claim 18 , wherein the means for detecting the secondary antibody is hydrogen peroxide or streptavidin. 
   
   
       22 . A method for diagnosing an attack of disease which disrupts erythrocyte by using a kit for immunodetection of heptoglobin of  claim 18 , which comprises the steps of:
 (i) collecting blood sera from a normal and a subject who is doubted as a patient suffering from a disease which disrupts erythrocyte;   (ii) adding each of the collected sera to a plate in the said kit to react with an antibody fixed on the plate;   (iii) removing the serum from the plate and adding a solution containing an antibody in the said kit to the plate;   (iv) removing the solution from the plate and adding a secondary antibody in the said kit;   (v) removing the secondary antibody from the plate and adding a means for detecting the secondary antibody in the said kit to measure levels of heptoglobin; and,   (vi) comparing the levels of dimeric heptoglobin with each other.   
   
   
       23 . The method for diagnosing an attack of disease which disrupts erythrocyte of  claim 22 , wherein the disease is malaria. 
   
   
       24 . The method for diagnosing an attack of disease which disrupts erythrocyte of  claim 22 , wherein the secondary antibody is labeled with biotin- or peroxidase-labeled rabbit-goat serum. 
   
   
       25 . The method for diagnosing an attack of disease which disrupts erythrocyte of  claim 22 , wherein the means for detecting the secondary antibody is hydrogen peroxide or streptavidin. 
   
   
       26 . A kit for diagnosing malaria, which comprises:
 (i) a plate on which an antibody which binds specifically to α subunit or β subunit of heptoglobin is fixed;   (ii) a solution containing an antibody which binds specifically to α subunit or β subunit which is not bound to the antibody fixed on the plate;   (iii) biotin- or peroxidase-labeled secondary antibody which binds to the antibody in the solution; and,   (iv) hydrogen peroxide or streptavidin for detecting the secondary antibody.   
   
   
       27 . The method for measuring heptoglobin level in blood serum of  claim 5 , wherein the labeling of antibody is done by fluorescent material, radioisotope or enzyme. 
   
   
       28 . The method for diagnosing an attack of disease which disrupts erythrocyte of  claim 13 , wherein the labeling of antibody is done by fluorescent material, radioisotope or enzyme.